(1) Field of the Invention
This invention generally relates to the field of diagnostic virology and, more particularly, to a method for detecting infectious herpesvirus in a specimen and a cell line for use therefor.
(2) Description of the Related Art
Herpesviruses have been found in most animal species and approximately 100 herpesviruses have been at least partially characterized. These include species causing diseases in humans, horses, cattle, pigs, and chickens. In humans, the eight herpesviruses that have been thus far isolated are viewed as important causes of human morbidity and mortality. (Whitley, R. J., Virology (1990) New York, Raven Press which is incorporated by reference). Thus, the availability of methods for detecting infectious herpesviruses has become increasingly important.
The eight known human herpesviruses are herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), human cytomegalovirus (HCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpes virus 6 (HHV6), human herpes virus 7 (HHV7), and human herpes virus 8 (HHV8). The herpesviruses differ with respect to the diseases they cause. HSV-1 infection produces skin vesicles or mucosal ulcers generally limited to the oropharynx; HSV-2 produces mucocutaneous lesions generally in the genital region; HCMV can infect monocytes and a number of organ systems including salivary glands, kidneys, liver and lung; VZV causes the diseases of chickenpox and shingles; EBV infects the oropharyngeal epithelium and B lymphocytes; and HHV6 and HHV7 infect mononuclear cells and, in children, produce skin eruptions (Roseola Infantum). (Virology, Fields and Knipe, Eds., Raven Press, pp. 1795-2062, which is incorporated by reference).
All herpes virus have a linear double-stranded DNA genome and they all replicate in the nucleus of infected cells where viral gene expression during viral replication occurs as an ordered cascade. Genes expressed during viral replication are organized on the genome in a very straightforward manner; there are few overlapping genes, very few spliced genes, and the regulatory elements (for example, promoters) are immediately upstream of the open reading frames. All known herpes viruses have three major classes of herpesvirus genes, .alpha., .beta., and .gamma., which have the same basic temporal pattern of expression during viral replication.
Alpha genes, also called immediate-early genes, are expressed very early after infection and the expression of each alpha gene does not require any other viral gene or gene product. The products of the alpha genes are predominantly involved in regulation of viral gene expression.
Beta (early) genes are expressed only after the alpha genes because their expression depends on the presence of one or more of the alpha gene products which act as transcriptional activators to upregulate the expression of the beta genes. Thus, one way that beta genes have been defined is by the observation that their expression from the viral genome is not reduced in infected cells when viral DNA synthesis is blocked, but there is a virtual absence of expression of their RNA transcripts when production of alpha gene products is prevented by blocking protein synthesis. The products of beta genes are primarily enzymes involved in viral nucleic acid synthesis and metabolism.
Gamma (late) genes are expressed either primarily (.gamma.1) or exclusively (.gamma.2) following viral DNA synthesis. Gamma gene products are primarily structural components of the virion.
Many studies have analyzed the regulation of herpesvirus gene expression using isolated herpesvirus genes or isolated herpesvirus promoters, outside the context of the viral genome. This experimental approach has contributed to the identification of the cis- and trans-acting factors involved in the regulation of the expression of many herpesvirus genes. Many studies have shown that isolated beta genes, such as the prototypical beta gene, the HSV thymidine kinase (tk) gene, when transfected into cells are capable of being upregulated by certain alpha genes (Eisenberg et al., Mol. Cell Biol. 5:1940-1947, 1985) which is incorporated herein by reference). The tk promoter has been shown to contain cis-acting elements that are found on many cellular promoters (Zipser et al., Proc. Natl. Acad. Sci USA 78:6276-6280, 1981, incorporated herein by reference). It is generally recognized that the promoter of beta genes is both necessary and sufficient for the transactivation of beta genes by alpha gene products.
The complete sequences of human herpesviruses HSV-1, HSV-2, HCMV, EBV, VZV, HHV6, HHV7 are known, and a partial sequence of HHV8 is known. (HSV-1, McGeoch et al., Nucleic Acids Res 14:1727-1745, 1986, McGeoch et al., J Gen Virol 69:1531-1574, 1988, GenBank Accession Nos.: X14112, D00317, D00374, and S40593); HSV-2, McGeoch et al., J. Gen. Virol. 72, 3057-3075, 1991, GenBank Accession No.: Z86099; HCMV, Chee et al., DNA Seq 2:1-12, 1991, GenBank Accession No.: X17403; EBV, Baer et al., Nature 310:207-211, 1984, GenBank Accession Nos.: V01555, J02070, K01729, K01730, V01554, X00498, X00499, and X00784; VSV, Davidson and Scott, J. Gen Virol 67:1759-1816, 1986, GenBank Accession Nos.: X04370, M14891, and M16612; HHV6, Gompels et al., Virology 209:29-51, 1995, GenBank Accession No.: X83413; HHV7, Nicholas, GenBank Accession No.: U43400; and HHV8, Russo et al., Proc. Natl. Acad. Sci. U.S.A. 93:14862-14867, 1996, GenBank Accession No.: U75698, each of which is incorporated by reference). Analysis of this sequence data has shown that the beta genes represent a limited number of genes in the genomes of all herpesviruses which have been studied and that beta genes are highly conserved in the herpesvirus family.
For example, in HSV-1 there are fourteen genes that have been classified as beta genes: UL2, UL5, UL8, UL9, UL12, UL23, UL29, UL30, UL39, UL40, UL42, UL50, UL52, and UL53 (Roizman et al., Herpes Simplex Viruses and Their Replication, Raven Press, Ltd. NY, pp. 1795-1841, 1990, incorporated herein by reference). These genes encode respectively, a uracil DNA glycosidase, a DNA helicase, a component of the DNA helicase/primase complex, an origin of DNA replication binding protein, a DNA exonuclease, a nucleoside kinase, a single-stranded DNA binding protein, a DNA polymerase, a ribonucleotide reductase large subunit, a ribonucleotide reductase small subunit, a double-stranded DNA binding protein which acts as a polymerase processivity factor, a dUTPase, a primase, and a protein kinase. All but one of these enzymes, the protein kinase, has been shown to be involved in DNA metabolism or to be directly involved in synthesis of viral DNA.
Based on standard DNA and predicted protein sequence alignment paradigms, it has been determined that HSV-2 and VZV have homologs for each of the fourteen HSV-1 beta genes (Davison et al., J. Gen. Virol. 67:1759-1816 1986, incorporated herein by reference). For example, the UL39 gene of HSV-1 encodes the large subunit of ribonucleotide reductase (RR), a two subunit enzyme involved in the generation of deoxyribnucleoside triphosphates, the immediate precursors of DNA. The ribonucleotide reductase large subunit of HSV-1, also known as RR1 or ICP6, has 38% homology at the N-terminal portion and 93% homology at the C-terminal portion of the corresponding HSV-2 protein, ICP10, which is encoded by the UL39 gene of HSV-2. (Nikas et al., PROTEINS: Structure, Function, and Genetics 1:376-384, 1986 which is incorporated by reference). In VZV, the corresponding ribonucleotide reductase large subunit is encoded by gene 19 and shows between 43% and 53% homology beginning at HSV-1 amino acid 384 and VZV amino acid 16. Id. Homologs to UL39, gene 19 and most of the other HSV and VZV beta genes have also been easily identified in more distantly related human herpesviruses such as Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) (Baer et al., Nature 310:207-211, 1984; Chee et al., Curr. Top. Microbiol. immunol. 154: 125-169, 1990, each of which is incorporated herein by reference). A listing of the homologous beta genes in the human herpesvirus family is shown in FIG. 20.
In those cases which have been studied, the products of these conserved genes have displayed remarkable conservation of function and all, except for the protein kinase gene, have been shown to have a role in viral DNA synthesis or metabolism. Moreover, in all cases studied, these genes exhibit a pattern of expression consistent with their being classified as beta genes.
Herpes simplex viruses Types 1 and 2 (hereinafter referred to collectively as HSV) infect a large number of individuals each year. Primary infection of immunocompetent patients with HSV usually leads to a mucocutaneous syndrome such as herpes labialis (HSV-1) or herpes genitalis (HSV-2), the latter being one of the most common sexually transmitted diseases today. Infection with HSV can also cause more serious infections, the most serious of which are sight-threatening keratitis and life-threatening encephalitis. Moreover, HSV related disease in immunocompromised individuals such as newborns, leukemia patients, organ transplant recipients and AIDS patients has become an increasingly prevalent and difficult problem.
Significant advances have been made in the treatment of herpesvirus infections in the past decade. These advances in antiviral therapy have expanded the role of the diagnostic virology laboratory and have identified the need for more sensitive, accurate and rapid diagnostic tests to assist in the early diagnosis of herpesvirus infections.
Various tests are presently available for the diagnosis of HSV infections. Most involve the detection of viral antigens or intact infectious virus. Antigen detection assays offer the advantage of rapidity and specificity, but can lack the necessary sensitivity. Kowalski, R. P. and Gordon, Y. J., Ophthal. 96:1583-1586 (1989). The most reliable test to detect infectious herpesvirus involves inoculation of specimens onto tissue culture cells followed by detection of infectious virus by microscopically observing a characteristic cytopathic effect. Although HSV is a relatively easy virus to culture as it replicates on a wide variety of continuous cell lines, virus propagation in tissue culture can be slow and expensive. Recently, improved techniques have been developed for the detection of viruses from clinical specimens. The shell vial technique, for instance, has greatly increased the sensitivity and the rapidity of HSV detection. When this method is combined with antigen detection by immunohistochemistry, HSV can be positively identified within 24 hours in the majority of cases. Gleaves et al., J. Clin. Micro. (1985) 21:29-32; Ziegler et al., J. Clin. Micro. (1985) 26:2013-2017. While this type of assay is preferred in diagnostic virology applications, it is labor intensive and a significant number of specimens are not identified as positive until after 48 hours. Another recent technological advance, polymerase chain reaction (PCR) technology, presents a promising tool for the detection of HSV particularly in cerebrospinal fluid specimens, but this technology detects viral nucleic acid and not infectious virus. Puchhammer-Stockl, et al., J. Med. Virol. (1990) 32:77-82. The detection of infectious virus is often preferred because it definitively indicates that there is an ongoing viral infection with active viral replication. PCR detection of viral nucleic acid may only be indicative of the presence of a remnant of a past infection or the presence of a latent infection.
Previous scientific studies involving herpesviruses have used susceptible cell lines transfected with a chimeric DNA construct containing a marker gene in transient assays to study various aspects of the virus such as the regulation of gene expression during viral replication. Flanagan W. M. and Wagner, E. K., Virus Genes 1:1:61-71 (1987). These studies have not, however, described a DNA construct stably integrated into the chromosome of a stable cultured cell line which is suitable for the diagnostic detection and quantification of a herpesvirus in a specimen with the requisite sensitivity and specificity for a clinical diagnostic assay.
Recently, a method for detecting infectious HIV in a specimen has been disclosed that utilizes a genetically engineered cell line containing a chimeric gene having the E. coli LacZ gene associated with the HIV-1 LTR promoter. Rocancourt, et al., J. Virol. (1990) 64:2660-2668; Kimpton, J. and Emerman, M., J. Virol. (1992) 66:4:2232-2239. Although these cell lines may be useful for detecting HIV in a specimen, they are not suitable for diagnostic virology assays because of their lack of specificity. It is well-known that the HIV-1 LTR promoter used in the DNA construct of these studies to cause expression of the reporter gene is not specific for HIV and that other viruses cause expression of the reporter gene if present in the specimen. In particular, the presence of HSV or cytomegalovirus in the specimen causes activation of the LTR promoter and subsequent expression of the reporter gene even in the absence of HIV in a specimen. Mosca, J. D., et al., Nature (1987) 325:67-70; Mosca, J. D., et al., Proc. Natl. Acad. Sci. (1987) 84:7408-7412; Popik and Pitha, Proc. Natl. Acad. Sci. (1981) 88:9572-9577. If such a cell line were used in a diagnostic assay, it could lead to the erroneous diagnosis of the presence of HIV in a specimen when in fact the specimen contained a different virus. Thus, the lack of specificity in cell lines prepared to detect HIV in a specimen prevents their use in a diagnostic assay which requires specificity.
A need exists, therefore, for a method for detecting a herpesvirus in a specimen that provides rapid detection in a cost efficient manner, while also providing the sensitivity and specificity necessary for a diagnostic assay.